Several photoreceptor-specific genes are actively transcribed in Y79 retinoblastoma (Rb) cells, making this cell line potentially useful for the study of photoreceptor metabolism. The utility of these cells is limited because commonly used methods of gene transfer into Y79 cells are inefficient and lack reproducibility. In contrast, we found that adenovirus transduction yields high efficiency gene transfer, however, generation of recombinant adenovirus is lengthy and time consuming. Here, we show that adenofection, a method of coupling adenovirus to plasmid DNA for improved gene transfer, is efficient for gene delivery into Y79 cells. Recombinant adenovirus expressing bacterial lacZ was noncovalently complexed to GFP or luciferase reporter plasmids with polyethylenimine. Efficiency of plasmid gene delivery was determined by monitoring GFP fluorescence. For comparison, calcium phosphate-mediated or cationic lipid transfection was performed in Y79 and HEK293 cells using standard protocols. The adenofection protocol yielded significantly higher efficiencies in Y79 cells than that obtained in these cells with calcium phosphate or cationic lipids. This method will facilitate any experiment requiring reproducible high-level gene transfer. Here, we show that adenofection can be used to analyze activity of the rod photoreceptor PDE6A gene promoter. © 2001 Elsevier Science B.V.